Myo-Inositol, hexakis(dihydrogen phosphate), dodecasodium salt

Endpoint summary

Description of key information

Not skin sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18.09.2017-13.10.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read across is given in section 13 of IUCLID.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

LuSens Assay submitted by BASF SE

Deviations:

yes

Remarks:

LuSens line was used; study was perfomred according to BASF SE protocol

Principles of method if other than guideline:

Deviations from the Guideline
The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is only based on the OECD 442D Guideline.
The deviations from the OECD 442D are given below:
1.For the test the LuSens cell line was used. This cell line was developed by the BASF SE.
2.In order to determine the concentration range applicable for experiment I and II a Cy-totoxicity Range Finder Test (CRFT) was performed. This test was performed in ac-cordance to the protocol of the BASF SE.
3.The dilution factor in the experiments is 1.2fold.
4.The controls are tested at only one concentration.
5.As positive control p-Phenylenediamine was used.
6.During the experimental performance the luciferase induction is measured at a second 96-well plate in accordance to the protocol of the BASF SE.
7.Regarding the acceptance criteria, the positive control must induce a luciferase induc-tion of a minimum of 2.5 fold in comparison to the solvent control. In addition the via-bility must be ≥ 70 %. The negative control must induce a luciferase induction of <1.5 fold and a viability of ≥ 70 %. Regarding the test item, a minimum of 3 test item con-centrations has to be analysable (viability: ≥ 70 %).
Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LU-CIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.
For this reason, all deviations of the LuSens test in comparison to the OECD 442D are declared as uncritical. The end result is not affected by those changes.

The deviations were assessed and signed by the study director on 25. Oct. 2017.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

According to Comission Regulation 2016/1688 amending Annex VII to regulation 1907/2006

Details on the study design:

VEHICLE CONTROL: Medium No. 3 (Biochrom GmbH)
POSITIVE CONTROL: p - Phenylenediamine
NEGATIVE CONTROL: D,L- Lactic acid
TEST SYSTEM:
- Cells used: LuSens Cell Line
- Supplier: BASF SE;Ludwigshafen, Germany
Preparation of cells:
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement).

PRELIMINARY STUDY:
-Cytotoxicity Assay
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. This yellow tetrazole is reduced to purple formazan in viable cells and can therefore be used for assessing of the cell metabolic activity. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the CRFT 12 nominal concentrations (0.2µg/ml - 400µg/ml) of the test item were tested.
The prepared cell plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h and 15 min.
After the incubation time the medium was removed from the cells. Afterwards, 200 μL of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Twelve wells were used as solvent control, 6 wells were used as growth control, 3 wells were used as negative control and 2 wells were used as positive control. The plate was sealed with breathable tapes to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards, the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the viability assay the MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution was added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards, the solution was removed and 100 μL MTT-lysis buffer was added to each well. The plate
was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer.
For calculation of the relative viability a validated Microsoft Excel® file was used. According to the results the dose levels for the
main study were selected
MAIN STUDY:
The chemicals must be tested in two independents experiments at least.
Experiment I and II were performed in the same way. Both prepared cell plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere
for 24 h and 30 min in Experiment I and 24 h and 45 min in Experiment II.
The treatment procedure was performed on both 96 well plates identically:
After the incubation time the medium was removed from the cells and 200 μL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile
compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.

- VIABILITY
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 μL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-
Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability

- LUCIFERASE INDUCTION:
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature.
During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.
For calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel® file was used.

Test positive Criteria:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is above or equal to 1.5 fold compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic tested concentrations whereby at least three tested concentrations must be non-cytotoxic.
Test negative Criteria:
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the above effects are not observed.

Positive control results:

Luciferase induction: 6.4/ 7.0 fold to solvent control
Relative Viability: 84.7/ 80.7%

Key result

Run / experiment:

other: 1

Parameter:

other: Luciferase induction

Value:

0.416

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Luciferase induction

Value:

0.394

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptability:
1. The average induction of the positive control was >= 2.5 and had a relative viability of >=70%
2. The induction triggered by the negative control and growth control was < 1.5 fold compared to the induction of the solvent control
and the viability was >= 70%
3.The average percentage standard deviation (luciferase induction) of the variability in at least21 solvent control wells were below 20 %.
4. More than 3 concentrations have been within viability limits, i.e. have relative viability of at least 70 %.
Thereby the acceptability (validity) criteria have been fulfilled

RESULTS

- Results of Experiment I

All control substances indicated the expected effect.

No considerable reduction of the viability was detected (all values ≥ 84 %).

Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 6.4 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 54 μg/mL to 333 μg/mL.

The viability values were all ≥ 70 % and therefore analysable for luciferase induction.

A slight cytotoxic effect was only observed at the highest concentration 400 μg/mL. Therefore, the result of this concentration is not included for the final evaluation.

In the Luciferase assay, none of the tested non cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Results of Experiment II

All control substances indicated the expected effect.

No considerable reduction of the viability was detected (all values ≥ 80 %).

Regarding the Luciferase induction, the growth control

and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 7.0 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 54 μg/mL to 278 μg/mL.

The viability values were all ≥ 76 % and therefore analysable for luciferase induction.

Slight cytotoxic effects were only observed at the two highest concentrations 400 μg/mL and 333 μg/mL. Therefore, the results of those concentrations are not included for the final evaluation.

In the Luciferase assay, none of the tested non cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate
the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (400 μg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.21) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

Vehicle control (Medium no. 3), negative control ( Lactic acid) and postive control (p - Phenylenediamine) run in parallel

No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test

item.

Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Justification for read across is given in section 13 of IUCLID.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Justification for non-LLNA method:

According to Comission Regulation 2016/1688 amending Annex VII to regulation 1907/2006

Key result

Run / experiment:

other: 1

Parameter:

other: Luciferase induction

Value:

0.416

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Luciferase induction

Value:

0.394

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate
the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (400 μg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.21) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

Vehicle control (Medium no. 3), negative control ( Lactic acid) and postive control (p - Phenylenediamine) run in parallel

No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test

item.

Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Following the read across justification this result can be used as weight of evidence approach to justify the non classification for "Esters of sodium phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), dodecasodium salt).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05.02.2018-08.03.2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study as required by REACH regulation 1907/2006
Justification for read across is given in section 13 of IUCLID.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Method No. 442C, adopted 04. Feb.2015: “In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)“
EURL ECVAM (European Union Reference Laboratory for alternatives to animal testing):
“DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitation
Testing.”, 29. Jun. 2015

Deviations:

yes

Remarks:

Deviation in Cys - peptide stock and calibration solution. This was considered uncritical because the deviation was < 0.2 % from the nominal values and therefore negligible.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to Comission Regulation 2016/1688 amending Annex VII to regulation 1907/2006

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No: NA2253
- Expiration date of the lot/batch: June 2019
- Purity test date: June 2016

Details on the study design:

VEHICLE:
POSITIVE CONTROL:
- Cysteine peptide: cinnamaldehyde
- Lysine peptide: 2,3 Butanedione

SOLVENT CONTROLS:
- Cysteine - and Lysine peptide: Acetonitrile replacing the test item stock solution

Co - ELUTION CONTROL:
Sample prepared from the respective peptide buffer and the test item, but without peptide

NEGATIVE CONTROL

TEST SYSTEM
Peptides with ≥ 95 % purity,.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Supplier/Manufacturer: Synthesized by Genecust, Dudelange, Luxemburg

CONDUCTION OF THE STUDY
1. DISSOLUTION OF THE TEST ITEM
As the test item is a UVCB, a saturated solution prepared from a filtrated 100 mg test item/mL suspension will be prepared and used as 100 mM solution. 50 μL test item per 1 mL assay for the cysteine peptide and 250 μL test item per 1 mL assay for the lysine peptide are used, the residual volume is filled with acetonitrile (equivalent to 1:10 and 1:50 ratio, respectively).
2. PREPARATION OF TEST SOLUTIONS:
- Dilution buffer
All volumes are exemplary and may be scaled to the amount required, maintaining the ratios.
20 mL Acetonitrile is mixed with 80 mL phosphate buffer, pH 7.5 (Peptide dilution buffer C)
20 mL Acetonitrile is mixed with 80 mL ammonium acetate buffer, pH 10.2 (Peptide
dilution buffer K)
- Peptide stock solutions
Peptide stock solutions are freshly prepared for each assay.
0.667 mM Cys-Peptide solution is prepared by dissolving an appropriate amount of the peptide in 25 mM phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution is prepared by dissolving an appropriate amount of the peptide in 25 mM ammonium acetate buffer, pH 10.2.
- Test item stock solution
A saturated test item solution is freshly prepared for each assay using 100 mg/mL nominal concentration in acetonitrile. 300 mg test item is sonicated in 3 mL acetonitrile and filtrated over 0.45 μm PTFE membrane syringe filter for use in the assay. The filtrate is treated as 100 mM stock solution.
- Peptide calibration standards
From each peptide stock solution the following calibration standards will be prepared in the appropriate dilution buffer : 0.534 / 0.267 / 0.134 / 0.067 / 0.033 /
0.017 mM peptide. Blank dilution buffer is also measured. Calibration samples are analysed before the samples containing the test item.
- Test item samples
Samples are prepared in triplicate for each peptide. The Cys-peptide samples are prepared in 1:10 ratio (1 mL assay size with final concentration 0.5 mM peptide:50 μL test item), the Lys-peptide samples in 1:50 ratio (1 mL assay size with final concentration 0.5 mM peptide:250 μL test item) using the stock solutions
INCUBATION
The positive control, solvent control sets C, and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ±2.5 °C for 24 ± 2 h. Samples appearing turbid or where precipitation is visible by the unaided eye are centrifuged (benchtop centrifuge, 10 min at 4500 rpm) and only the clear supernatant is used for measurement.

ANALYTICAL MEASUREMENT
Analytical instrument: HPLC system
Column : ACE Excel SuperC18 150x3 mm column with 3 μm particles
UV/VIS-Detector
HPLC program:
Eluent A H2O + 0.1 % TFA
Eluent B Acetonitrile + 0.085 % TFA
time (min) % A % B
0 90 10
10 75 25
10.5 10 90
12 10 90
13 90 10
20 90 10
Flow rate: 0.55ml/min
Injection volume 7 μL
Column temperature 30 °C
Wavelength 1 220 nm; Wavelength 2 258 nm
CRITERIA:
Acceptance Criteria postive controls:
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion
Acceptance criteria solvent control samples
The measured values of solvent control samples of set A should be 0.5 ± 0.05 mM
The mean peptide concentration of the three reference controls C in the solvent used for the test item stock solution should be 0.50 ± 0.05 mM.
The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
EVALUATION OF RESULTS
As no prediction model exists for UVCB substances, the Cysteine 1:10/lysine 1:50 prediction model is used. According to the test guideline, the reactivity is classified as “high”, “moderate”, “low” or “minimal” using the Cysteine 1:10/Lysine 1:50 prediction model

Positive control results:

The acceptance criterie for the two positive results are met. Three replicates were measured.
1. Cinnamaldehyde (pos. control for Cys-peptide): Mean value (n=3): depletion [%] 81.40 SD 2.00
2. 2,3, Butanedione (pos. control for Lys-peptide): mean value (n=3): Depeletion [%] 26.01 SD 2.69
Results are within the ranges of the acceptance criteria.

Key result

Run / experiment:

other: Depletion values for Lys - Peptide

Parameter:

other: Peptide Depeletion [%]

Value:

0.78

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: depletion values for CYS - Peptide

Parameter:

other: peptide depletion [%]

Value:

1.83

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The standard calibration curve should have an r² > 0.99
The measured values of solvent control samples of sets A and C should be 0.5 ± 0.05 mM
The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.

Proficiency:

The ten proficiency chemicals listed in the guideline were tested using the HPLC analysis method. Eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline (LAUS reference study 201704R875).

Interpretation of results:

GHS criteria not met

Conclusions:

The DPRA predicition is “negative” with minimal reactivity

Executive summary:

The study was performed according to OECD guideline 442c in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. As the test item is a UVCB substance, no molecular weight is known and therefore a saturated test item solution in acetonitrile was used instead of a 100 mM solution and incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively. The peptide concentration after incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio (calculated treating the saturated test item solution as 100 mM stock solution) of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel. As no prediction model exists for UVCB substances, the Cysteine 1:10/lysine 1:50 prediction model is used.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 1.31% therefor the test item was classified with:

DPRA predicition is “negative”

Reactivity class: "minimal"

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Justification for read across is given in section 13 of IUCLID.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Key result

Run / experiment:

other: Depletion values for Lys - Peptide

Parameter:

other: Peptide Depeletion [%]

Value:

0.78

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: depletion values for CYS - Peptide

Parameter:

other: peptide depletion [%]

Value:

1.83

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The DPRA predicition is “negative” with minimal reactivity

Executive summary:

The study was performed according to OECD guideline 442c in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. As the test item is a UVCB substance, no molecular weight is known and therefore a saturated test item solution in acetonitrile was used instead of a 100 mM solution and incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively. The peptide concentration after incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio (calculated treating the saturated test item solution as 100 mM stock solution) of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel. As no prediction model exists for UVCB substances, the Cysteine 1:10/lysine 1:50 prediction model is used.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 1.31% therefor the test item was classified with:

DPRA predicition is “negative”

Reactivity class: "minimal"

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Following the read across justification this result can be used as weight of evidence approach to justify the non classification for "Esters of sodium phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), dodecasodium salt).

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13.03.1995-21.04.1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Study was performed before the REACH regulation 1907/2006 came into force.
Justification for read across is given in section 13 of IUCLID
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

This method is similar to that described in the Official Journal of the European Communities No. L383 A131 B.6. Acute Toxicity (Skin Sensitization), 29.12.1992 and OECD Guideline 406 for Testing of Chemicals (1993).

Deviations:

not specified

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In - vivo test was performed in 1995

Specific details on test material used for the study:

The test material is a 50% aqeous solution of Esters of phosphoric acid with myo-Inositol, plantderived (old name: Fytic acid), CAS 83-86-3 which is the commonly available quality.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Albino Dunkin Hartley strain guinea pigs were obtained from Harlan Olac.
On receipt, the animals were earmarked uniquely and held in the holding area for 1 day.
Housing: The animals were housed in groups of four in stainless steel cages
Diet: Fed pelleted diet (FD1, Special Diet Services)
Water: Ad libitum.
The animal room was air conditioned to maintain temperatures at 22± 3°C
Relative humidity at 50% +1- 20%.
The room lighting was controlled to provide 12 hours light and 12 hours dark in each 24 hour cycle.

Route:

intradermal

Vehicle:

other: physiological saline (Ringer)

Concentration / amount:

0.1%/ 0.1 ml

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: physiological saline (Ringer)

Concentration / amount:

5,0 % a.i. (w/v)

Day(s)/duration:

48 h

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

2.5% a.i. (w/v)

Day(s)/duration:

24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 (10 male/ 10 female) were randomly selected for the test group
10 (5 male/ 5 female) were randomly selected for the control group

Details on study design:

TEST SOLUTIONS:
A. INDUCTION PHASE
- Test solutions for injection:
1) A 1:1 mix of Freund`s Complete Adjuvant (FCA) with 0.9% (w/v) physiological saline (saline).
2) 0.1 % a.i. (w/v) phytic acid in saline.
3) 0.2 % a.i. (w/v) phytic acid in saline mixed 1:1 with FCA to give a final concentration of 0.1 % a.i. (w/v) of phytic acid.

- Test solution for occlusive patch
5.0% a.i. (w/v) phytic acid in saline.

- Control solutions (Injection)
1. 1:1 FCA mix. and saline.
2. saline.

B. CHALLENGE PHASE
- Test solution for occlusive patch
2.5% a.i. (w/v) phytic acid in saline.

TEST METHOD:
INDUCTION PHASE
Induction consisted of two stages, intradermal injection followed seven days later by
occluded patch application.
- Intradermal injection
The hair was clipped from a 2cm x 4cm area of skin in the dorsal shoulder area and three
pairs of intradermal injections (test solution 1-3 / control solutions 1-2-1) were made within the clipped area
- Occlusive Patch
Seven days after the injections the same 2cm x 4cm area was clipped and shaved. A 2cm x 4cm filter paper patch, attached by double-sided adhesive tape to a 4cm x 6cm piece of thin polythene, was saturated with 5.0% a.i. phytic acid in saline and placed over the shaved site. The patch was held in place for 48 hours by adhesive plaster wrapped around the trunk behind the forelimbs.
Control group received an occluded patch of the test solvent over the injection sites.
The occluded patch site was scored 24 hours after removal of the bandage
- Challenge
Fourteen days after the application of the induction patch the guinea pigs of both groups were challenged on the clipped and shaved flank by occluded patch. For each animal, an 8mm diameter filter paper patch in an llmm aluminium patch test cup was saturated with 2.5% a.i. phytic acid in saline and the patch applied to the shaved flank. The patch was held in place for 24 hours by adhesive plaster wound around the trunk. The treatment sites were examined for evidence of sensitization 24 and 48 hours after removal of the patches

Challenge controls:

10 control guinea pigs were selected with similar body weights to those of the test guinea pigs at that challenge.
The guinea pigs received two intradermal injections of 1:1 mixture of FCA and saline, two O.l ml injections of saline and two O.l ml injections of a 1:1 mixture of
FCA and saline in exactly the same way as the test animals.
This was followed seven days later by a 24 hour occluded patch of the test solvent over the injection sites. After both of the induction procedures the animals were assessed in exactly the same way as the test animals.
At challenge the guinea pigs were challenged with the test material in exactly the same way as the test animals.

Positive control substance(s):

yes

Remarks:

Hexyl Cinnamic Aldehyde (periodic tests are run at the laboratory)

Positive control results:

As a check on the sensitivity of the method carried out in ESL, one positive control material was subjected to the normal M&K test procedure at intervals specified below.
Summary data for a period relating up to and including the study of phytic acid were: HEXYL CINNAMIC ALDEHYDE
Concentrations:
Induction injection 0.5% (v/v)
Induction application 50.0% (v/v)
Challenge application 10.0% (v/v)

Results of tests
Study number Date % positive
SM930149 06 April 1993 80%
SM930150 23 November 1993 80%
SM940210 09 May 1994 70%
SM940414 01 November 1994 70%

For study number SM930150 the following concentrations were used:
Concentrations :
Induction injection 1.0% (w/v)
Induction application75.0% (w/v)
Challenge application 5.0% (w/v)

For study number SM940210 and SM940414 the following concentrations were used:
Concentrations: Induction injection
Induction application
Challenge application
1.0% (w/v)
50.0% (w/v)
10.0% (w/v)

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

1

Total no. in group:

9

Clinical observations:

10 animals were selected for the control group but one animal was found dead after induction application phase

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

10 animals were selected for the control group but one animal was found dead after induction application phase

Remarks on result:

no indication of skin sensitisation

Reading:

other: positiv control standard test

Hours after challenge:

48

Group:

positive control

Dose level:

10% (v/v) Hexyl Cinnamic Aldehyde

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

test is periodically performed at the laboratory

Preliminary irritation tests

It was not considered necessary to adjust the pH of the test material for the sensitization test as the results of the preliminary irritation tests showed that the irritancy of phytic acid was not significantly altered and the concentrations selected would be the same.

As a result of the preliminary irritation tests, 0.1 % a.i. was selected from the preliminary irritation test to be the highest suitably irritant concentration for the intradermal injection

induction. From the preliminary occluded patch test, 5.0% a.i. was selected as it was the highest suitably irritant concentration for the patch induction. 2.5% a.i was selected as

the highest non-irritant concentration for the challenge patch. .

Sensitization test

The test and control guinea pigs showed intense (grade 3) erythema reactions to the induction injection and induction application treatments.

There was no evidence of sensitization reactions in any of the twenty test guinea pigs challenged with 2.5 % phytic acid at challenge 1.

One control animal showed a minor irritation reaction to the test material at the 24 hour assessment, the rest of the control animals showed no response. One test animal showed a minor irritation reaction to the test material at the 24 hour assessment.

The results are summarised in Table 1.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, phytic acid was classed as a non-sensitiser

Executive summary:

The study following the principles of OECD 406 (1993) (Magnusson & Kligman Maximization test) was performed to determine the potential of phytic acid to induce skin sensitization reactions.

Sensitization was induced in guinea pigs by intradermal injections of both test substance and Freund Is Complete Adjuvant. Seven days later the induction process was supplemented by application of the test substance to the shoulder injection sites under occlusion for 48 hours. Fourteen days later the guinea pigs were challenged by a 24 hour occluded patch of the test substance, the treatment sites were then examined for evidence of sensitization 24 and 48 hours after removal of the challenge patch.

The test and control guinea pigs showed intense (grade 3) erythema reactions to the induction injection and induction application treatments.

There was no evidence of sensitization reactions in any of the twenty test guinea pigs challenged with 2.5 % phytic acid at challenge 1.

One control animal showed a minor irritation reaction to the test material at the 24 hour assessment, the rest of the control animals showed no response. Test animal . All animals showed a minor irritation reaction to the test material at the 24 hour assessment.

Under the conditions of this study, phytic acid was classed as a non-sensitiser.

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Study was performed before the REACH regulation 1907/2006 came into force.
Please refer to read across justification provided in Section 13.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Group:

negative control

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

1

Total no. in group:

9

Clinical observations:

10 animals were selected for the control group but one animal was found dead after induction application phase

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2.5% a.i. (w/v)

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

10 animals were selected for the control group but one animal was found dead after induction application phase

Remarks on result:

no indication of skin sensitisation

Reading:

other: positiv control test is performed periodically

Hours after challenge:

48

Group:

positive control

Dose level:

10% (v/v) Hexyl Cinnamic Aldehyde

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, phytic acid was classed as a non-sensitiser

Executive summary:

The study following the principles of OECD 406 (1993) (Magnusson & Kligman Maximization test) was performed to determine the potential of phytic acid to induce skin sensitization reactions.

Sensitization was induced in guinea pigs by intradermal injections of both test substance and Freund Is Complete Adjuvant. Seven days later the induction process was supplemented by application of the test substance to the shoulder injection sites under occlusion for 48 hours. Fourteen days later the guinea pigs were challenged by a 24 hour occluded patch of the test substance, the treatment sites were then examined for evidence of sensitization 24 and 48 hours after removal of the challenge patch.

The test and control guinea pigs showed intense (grade 3) erythema reactions to the induction injection and induction application treatments.

There was no evidence of sensitization reactions in any of the twenty test guinea pigs challenged with 2.5 % phytic acid at challenge 1.

One control animal showed a minor irritation reaction to the test material at the 24 hour assessment, the rest of the control animals showed no response. Test animal . All animals showed a minor irritation reaction to the test material at the 24 hour assessment.

Under the conditions of this study, phytic acid was classed as a non-sensitiser. Following the read across justification this result can be used as weight of evidence approach to justify the non classification for "Esters of sodium phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), dodecasodium salt).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a weight of evidence approach two studies (OECD 442D, 442C) have been performed with "Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt, CAS 14306 -25 -3) and one study (OECD 406) with 50% aqueous solution of the "Esters of phosphoric acid with myo-Inositol, plant-derived (old name: Fytic acid).

Under the condition of the in - vitro method OECD 442D (LuSens assay) the test item was negative and is therefore considered not having the potential to activate the Nrf2 transcription factor (=> no sensitizing potential).

Under the conditions of the in - chemico method OECD 442 C (DPRA) the prediction for the test item is "negative" with minimal reactivity

Under the conditions of the in-vivo method (OECD 406) fytic acid was classed as a non-sensitiser.

Justification for classification or non-classification

According to the CLP Regulation (EC) No. 1272/2008 3.4.2.2.1. a substance shall be classified as a skin sensitizer category I where data are not sufficient for sub-categorisation. Where data are sufficient, a refined evaluation according to section 3.4.2.2.1.3 allows the allocation of skin sensitisers into sub-category 1A, strong sensitisers, or sub-category 1B for other skin sensitisers.

The concludsion for non - classification of the substance "Esters of sodium phosphate with myo-Inositol, plant-derived" is based on the study results of OECD 442 C and OECD 442 D available for the structure analogue "Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt; CAS 14306 -25 -3) and the result of one study similar to OECD 406 available for the structure analogue "Esters of phosphoric acid with myo-Inositol, plant-derived" (old name: Fytic acid). A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13)

Considering the weight of evidence approach from data obtained from one in - vitro (OECD 442d) and one in - chemico (OECD 442c) study performed with one similar substance and anaother in - vivo study performed with another similar substance, it can be finally concluded that the substance "Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt; CAS 14306 -25 -3) does not meet the criteria for classification according to the CLP Regulation (EC) No.1272/2008 3.4.2.2.